值得关注的线粒体12SrRNA C1494T突变--药物敏感致聋靶点

A Noteworthy Mitochondrial DNA Mutation,12SrRNA C1494T-a Sensitive Molecular Site for the Drug-induced Hearing Loss

目的探讨线粒体12SrRNA C1494T突变与氨基糖苷类药物致聋的临床表型特征的相关性，警示临床值得关注的又一药物致聋敏感靶点。方法对遗传性聋资源库中两个具有母系遗传特征的耳聋大家系成员进行病史、体检、纯音测听及听性脑干反应检查并绘制系谱图。提取耳聋患者的外周血基因组DNA，运用聚合酶链扩增反应（polymerase chain reaction。PCR）方法对耳聋患者线粒体12SrRNA基因1228～1984位序列（涵盖12SrRNA 1494及1555位点）进行扩增，扩增产物纯化后进行限制性酶切鉴定或测序，运用DNAStar软件对测序结果进行比对分析。结果两个家系分别为一个四代相传的母系遗传家系（196家系）和一个三代相传的母系遗传性耳聋家系（1802家系）。196家系发现8名12SrRNA C1494T携带者，其中3名在接触氨基糖苷类药物后出现重度耳聋，成为聋哑人（Ⅱ：2、Ⅱ：5、Ⅲ：2）；2名无耳毒性药物用药史者（Ⅱ：9、Ⅱ：7），表现为高频听力损失，言语发育正常；1名患者25岁时应用链霉素后致重度耳聋，现交流困难（Ⅱ：3）；1名突变者现为2岁幼儿，目前对声音反应正常（Ⅳ：1）；1名为听力正常人（Ⅲ：3），现年28岁。1802家系发现12名耳聋患者，其中7名患者为母系后代，全部为聋哑，配偶聋哑人5名。1802家系中母系后代成员10名（3名男性，7名女性），其中7名为聋哑患者（2名男性，5名女性），3名为听力正常人（1名男性，2名女性）。7名母系后代患者中有4名有明确的耳毒性药物用药史（奎宁、链霉素或庆大霉素），3名用药史不详。2名母系患者进行基因检测发现线粒体12SrRNA C1494T突变。结论本研究通过两个线粒体12SrRNA C1494T突变家系警示线粒体12SrRNA C1494T突变又是一个值得非常关注的药物敏感作用靶点。具有此突变的患者接触氨基糖苷类药物会产生重度耳聋，而未接触该药物者可以是正常听力或是高频听力下降者。临床要高度警示线粒体12SrRNA C1494T突变患者，早发现、早预警可避免聋哑患者的出现。

Objective This study was to study the correlation between the clinical phenotype of patients with aminoglycoside-induced nonsyndromic sensorineural hearing loss and the mutation of mitochondrial DNA （mtDNA） 12SrRNA C1494T. Methods Two large Chinese families with maternally transmitted aminoglyeoside--in- duced hearing impairment were identified from the gene bank of inherited bearing disorders at Chinese PLA general hospital. All the members in the two families, extended from the probands, received detailed clinical evaluations ineluding medical history, general physical examinations, pure tone test and auditory brainstem response. Genomic DNA of the participants was extracted from the peripheral blood. The fragment spanning from mtDNA 12SrRNA position 1228 to 1984 （containing two known loci 1494 and 1555） was amplified by polymerase chain reaction （PCR）. The purified products were then subjected to direct sequencing or enzyn3e digestion. Finally, the sequence data were analyzed by using DNAStar software. Results The two families were a four--generation pedigree （pedigree 196） and a three generation pedigree （pedigree 1802）, with matrilineal inheritance of hearing loss respectively. Pedigree 196 had eight carriers for mtDNA 12SrRNA C1494T, of which three （Ⅱ-2, Ⅱ-5 and Ⅲ-2） exhibited severe sensorineural hearing impairment after exposure to an aminoglycoside antibiotic drug（s）, and finally progressed into deaf mutes. Two members （Ⅱ-9 and Ⅱ-7）, without any known medication of any aminoglycoside antibiotic drug showed high frequencies hearing loss, but their speech developed normally. Subject Ⅱ-3 had severe hearing impairment resulted from using streptomycin at age of 25 years, and now has difficulty in communication. Nevertheless, two subjects, a 2-years-old baby （Ⅳ-1） and a 28-years old adult （Ⅲ-3） showed no sign of hearing loss. Pedigree 1802 had 12 patients with hearing impairment. Seven matrilineal descendants were deaf mutes, and among the spouses, five were deaf mutes, too. There were a total of 10 matrilineal descendents （3 males and 7 females） in this pedigree, of which seven subjects （2 males and 5 females） were deaf mutes, and the remaining （1 males and 2 females） had normal hearing. Of the seven matrilineal descendents with hearing impairment, four pa- tients had clear history of using ototoxic drugs （e. g. Quinine, streptomycin and gentamycin）, while history of drnguse of other three subjects was not known. Genetic screening identified two matrilineal members carrying mtDNA 12SrRNA C1494T mutant. Conclusion Our results indicate that mtDNA 12SrRNA C1494T mutation is novel drug --sensitive molecular site imposed on Chinese population. These pedigree data show that individuals with C1494T mutation may develop severe hearing loss when exposed to ototoxic antibiotics, otherwise, they may have either normal hearing or high frequencies hearing loss. Therefore, these findings suggest the need for a preaution of the use of ototoxic antibiotics on the people who have mtDNA12SrRNA C1494T mutations to prevent patients with minor hearing loss from developing deaf mutes.